Project Summary. The separation of membranes into discrete compartments through the process of membrane fission is essential for diverse cellular processes ranging from cell division to viral entry. While the specialized fission machine dynamin is well-known to induce fission through constriction of membrane tubes, recent evidence shows that other proteins drive fission by previously unknown mechanisms. In particular, the epsin 1 N-terminal homology (ENTH) domain is a potent driver of membrane curvature (Ford et al., Nature 2002), and has recently been shown to play a role in membrane fission. Specifically, a recent report proposed that insertion of a wedge-like amphipathic helix by ENTH curves and destabilizes membranes, as evidenced by decreasing membrane fission ability among ENTH mutants with decreasing helix hydrophobicity (Boucrot et al., Cell 2012). However, our group recently showed that collisions among dense, membrane-bound ENTH proteins generate steric pressure, which drives membrane bending in the absence of helix insertions (Stachowiak et al., Nature Cell Biology 2012). These results prompted us to ask: is steric pressure also responsible for membrane fission by ENTH? In my preliminary studies, I found that ENTH mutants with reduced helix hydrophobicity are capable of driving fission to a similar degree as wild-type ENTH when bound to the membrane at comparable density. Interestingly, I also found that full-length epsin, which contains a bulky, intrinsically-disordered C-terminal domain, drives fission more potently than the ENTH domain alone. These results imply that, while helix insertions are important for binding proteins tightly to membrane surfaces, helices are not required for fission. However, once bound to the membrane surface at sufficient density, bulky molecules of arbitrary structure can create steric pressure that increases membrane curvature until fission occurs. Taken together, my findings reveal a novel mechanism for membrane fission. The objective of the proposed research is to quantitatively compare this new mechanism with other key mechanisms of membrane fission. The first specific aim will delineate the specific roles of wedge-like helix insertion and protein crowding in driving membrane fission. The second specific aim will examine how dynamin works cooperatively with helix insertion and protein crowding to drive robust fission. The third specific aim will utilize quantitative imaging of live cells to examine how helix insertion and protein crowding modulate fission dynamics in a physiological context. This work will create innovative biophysical tools for the simultaneous study of membrane fission and protein-lipid interactions both in vitro and in live cells. The overall outcome of this research will be a deeper understanding of the physical mechanisms of membrane fission, including the novel mechanism of membrane fission by protein crowding. The bold hypothesis described here asserts that any membrane-bound protein can contribute to fission, an idea that will influence understanding of diverse membrane compartmentalizing processes, including endocytosis, cell division, and viral entry.